Inflammatory bowel disease (IBD) is a group of common chronic disorders involving bowel inflammation. Ulcerative colitis (UC) and Crohn's disease (CD) are the most important conditions of this group. IBD is usually diagnosed in young adults. In most cases it is characterized by long remissions and incidental disabling flare-ups usually requiring treatment.
Currently there are about 2.4 million IBD patients in the EU and over 1.3 million in the US (Cosnes et al., 2011). The incidence and prevalence of the disease continue to grow (Molodecky et al, 2012). Once diagnosed, all IBD patients should be monitored for a possible relapse. Those developing relapses are treated, and treatment efficiency assessment is an important task in need of serious improvements. Differentiation between IBD and non-inflammatory diarrhoeas, such as Irritable Bowel Syndrome (IBS) constitutes another major problem, especially given that IBS affects 10-15% of the adult population in most developed Western countries (Agarwal & Whorwell, 2006; Khan & Chang, 2010).
IBD diagnosis confirmation usually requires colonoscopy, an efficient diagnostic procedure that is, however, highly invasive, expensive and can sometimes cause dangerous complications. Repeated colonoscopies in IBD patients may be dangerous; therefore currently IBD activity and therapy efficiency are usually assessed by disease activity indexes based on severity of clinical manifestations and results of indirect laboratory analyses (Satsangi et al, 2006). Colonoscopy is also widely used to differentiate between IBD and common functional conditions such as IBS.
Development of biomarker-based non-invasive diagnostic tests for IBD is generally recognised as an urgent problem, solution of which can help in addressing multiple important endpoints comprising: a) primary diagnosis of IBD and its differentiation from other conditions; b) differentiation between UC and CD; c) complication risk assessment; d) distinction between active IBD and remission; e) assessment of colonic mucosa damage and healing; f) IBD relapse prediction; g) Prediction of response to specific therapy and treatment choice; h) therapy efficiency monitoring and therapeutic adjustments (Lewis, 2011). Recent progress in biomarker use for these endpoints is reflected in numerous scientific publications and abundant patent literature. The main approaches can be roughly divided into methods employing non-colonic tissue or body fluids (especially blood/serum) and those using materials obtained directly from the colon, such as stool, colonic biopsy samples, and colonic lavage. The existing prior art is briefly outlined below.
The role of genetic factors in the pathogenesis of both UC (Louis et al, 2009) and CD (Weersma et al, 2009; Tamboli et al, 2011) is well known. There is a group of patent documents describing genetic markers for IBD diagnosis, often presented as complex multimarker panels exemplified by a family of patents by Harris and Alsobrook (U.S. Pat. Nos. 7,833,720; 7,833,721; 7,879,553; 7,923,544; 8,222,390; 8,227,589). Methods detecting single gene variants associated with IBD (WO03/052412) or specifically with CD (U.S. Pat. Nos. 6,001,569; 6,534,263) were also proposed. Determination of IBD-related changes in the human gut microbiome is the main element of another genotyping-based technique (US Pat. App. No. 2013/0045874). Other relevant patents using genetic markers describe approaches targeting changes in microRNAs (WO94/21662; US Pat App. Nos. 2011/0117111; 2013/0143764) and various gene expression profiles (U.S. Pat. Nos. 7,875,431; 8,257,923; US Pat. App. Nos. 2009/0155788; 2009/0186034; 2009/0311260; 2010/0267575; 2011/0082188; EP1462527). However, genetic approaches have not provided a clinically applicable method so far, being mostly confined to the prediction of Crohn's disease risk (Weersma et al, 2009) and assessment of pharmacogenetics of drugs used for its treatment (Roberts & Barclay, 2012). In addition to limited diagnostic efficiency, most of these methods are exceedingly technically complex.
The use of protein biomarkers defines another major group of original approaches to IBD diagnosis. Several existing patents describe diagnostic methods employing biomarkers of this type detectable in serum. Detection of perinuclear anti-neutrophil cytoplasmic antibodies (pANCA) associated with UC and CD-associated anti-saccharomyces cerevisiae antibodies (ASCA) constitutes an important component of most techniques using serum samples, however various additional markers were also considered (Peyrin-Biroulet et al, 2007). A number of multimarker methods proposed for human serum samples combines pANCA and/or ASCA with either multiple protein biomarkers of human or bacterial origin (U.S. Pat. Nos. 6,218,129; 7,608,414 7,759,079; 7,873,479; 8,315,818; 8,445,215; 8,463,553; US Pat. App. Nos. 2006/0154276; 2010/0015156; 2010/0021455; 2010/0129838; 2010/0254971; 2010/0255513; WO2005/009339) or additional genetic markers (US Pat. App. Nos. 2011/0229471; 2012/0171672; 2013/0203053; 2013/0225439). Some basically similar techniques for IBD detection using serum samples do not include pANCA or ASCA, being focused on other bacterial (U.S. Pat. Nos. 7,361,733; 7,993,865; 7,993,866; 7,993,867; 8,318,901; US Pat. App. Nos. 2007/0275424; 2011/0251100; WO2009/135257; WO2011/130546) or human (U.S. Pat. No. 7,358,058; US Pat. App. No. 2009/0258848) proteins associated with gut inflammation, in particular inflammatory cytokines (US Pat. App. Nos. 2010/0316992; 2012/0258883; WO2012/037199). In addition to ASCA, antibodies to GP2 (a membrane glycoprotein known to be expressed in the exocrine part of the pancreas) have recently been suggested as a new marker for CD (Somma et al, 2013). Other proposed techniques are designed to detect diagnostically informative subtypes of circulating monocytes (WO2012/172347), specific T-cell-associated molecules (U.S. Pat. No. 7,989,173) or goblet cell antigen elevated in UC patients (US Pat. App. No. 2008/0293625). In addition, methods determining oligosaccharide ratio changes in IgG (U.S. Pat. No. 8,043,832), assessing complex metabolite (over 100 small molecules) profiles (US Pat. App. No. 2012/0003158) or measuring antibodies against a range of dietary components (US Pat. App. No. 2012/0058497) were published. Although the outlined peripheral blood or serological marker panels may potentially be useful for differentiating UC from CD, disease monitoring and defining therapeutic strategies (Peyrin-Biroulet et al, 2007), they do not perform better than non-specific C-reactive protein (Palmon et al, 2008). None of them is currently applied for practical clinical use. Another major group of biomarker-based approaches in the area of IBD is related to analysing samples directly derived from the gastrointestinal tract. Prior art of this type deserves special attention since the present invention belongs to this group.
The idea of using colonic tissue for IBD testing could certainly be applied to invasively obtained tissue (biopsy) samples (U.S. Pat. No. 7,972,807; Us Pat. App. Nos. 2004/132110; 2009/0305267), but stool sample analysis appears to be the most frequently used approach. A range of marker proteins detectable in stool samples obtained from IBD patients was investigated in this context (reviewed by Foell et al, 2009 and Lewis, 2011). The principal candidates were proteins found in neutrophil granules, in particular calprotectin, lactoferrin, S100A12 protein, dimeric pyruvate kinase, polymorphonuclear elastase, myeloperoxidase and human neutrophil lipocalin (Foell et al, 2009; Lewis, 2011; Sherwood, 2012). Among them calprotectin detection in stool samples using ELISA assay developed and patented by Fagerhol et al (U.S. Pat. No. 5,455,160) was extensively investigated and provided the most consistent results (van Rheenen et al, 2010; Lewis, 2011). This calprotectin assay has recently been improved as described in US Pat. App. No. 2013/132347. A rapid calprotectin test for faecal samples has also been devised (WO2012/052586). Stool calprotectin quantification is the only biomarker-based test for IBD detection recommended for clinical use and currently employed by some clinicians (Sherwood, 2012).
Several patents by Boon et al describe IBD detection and differentiation from IBS using lactoferrin analysis in stool samples (U.S. Pat. Nos. 7,192,724; 7,560,240; 7,892,762). The same group also proposed methods for distinguishing between UC and CD that employed detection in faeces of already mentioned ASCA (U.S. Pat. No. 6,872,540) or pANCA (U.S. Pat. No. 7,736,858), alone or in combination with lactoferrin quantification (U.S. Pat. No. 7,785,818). In other publications lactoferrin test was combined with neopterin detection (US Pat. App. No. 2012/0258477) or with assays for several protein biomarkers comprising calprotectin and a group of interleukins (US Pat. App. No. 2011/0212104). At the same time lactoferrin quantification in stool was also proposed for colorectal cancer diagnosis (U.S. Pat. No. 5,552,292). In general IBD detection or differentiation between IBD and IBS using lactoferrin determination in faeces appeared to be less efficient than stool calprotectin assay (Sherwood, 2012).
Although information on S100A12 protein diagnostic performance for IBD detection is relatively scarce compared to calprotectin and lactoferrin, there are reports indicating that quantitative testing of stool samples for S100A12 may provide better results than calprotectin analysis (Kaiser et al, 2007), especially in children (de Jong et al, 2006; Sidler et al, 2008). Nevertheless, an attempt to use this marker for paediatric UC monitoring was not successful (Turner et al, 2010). In the absence of large clinical studies introduction of faecal S100A12 test into healthcare practice remains questionable (Sherwood, 2012). The lack of information on this biomarker is reflected in the available patent literature. The only relevant patent applications identified were US Pat. App. 2010/0311758 describing the use of S100A12 (alternatively called Calgranulin C) for diagnosing a wide range of inflammatory diseases and US Pat. App. No. 2009/0286328 proposing faecal S100A12 detection for colorectal cancer diagnosis. Dymeric pyruvate kinase (M2-PK), which was initially regarded as colorectal cancer marker detectable in stool samples (Hardt et al, 2004) has also emerged as a potential faecal marker for IBD (Jeffery et al, 2009; Turner et al, 2010). An ELISA assay for M2-PK is described in U.S. Pat. No. 5,972,628 and its variant for the protein detection in stool samples in U.S. Pat. No. 7,226,751. However, M2-PK is not applied in clinical practice.
Polymorphonuclear elastase is another enzyme present in neutrophils, which was proposed as a candidate IBD biomarker (Langhorst et al, 2008; Foell et al, 2009). Although an immunoassay for this protein exists (U.S. Pat. No. 6,124,107), it is not regarded as a potential clinical test.
Some authors also suggested that inflammation-related neutrophil degranulation can be detected in stool samples by quantifying myeloperoxidase (Wagner et al, 2008; Masoodi et al, 2011) and human neutrophil lipocalin (Nielsen et al, 1996, 1999), but these tests are not sufficiently studied to be proposed for IBD detection.
Proteins associated with eosinophils, such as eosinophil cationic protein and eosin-derived neurotoxin (EDN) can also be detected in stool, but they were usually described as faecal markers of intestinal hypersensitivity and eosinophilic inflammation (Foell et al, 2009). The eosinophil-derived neurotoxin (EDN, also called Eosinophil Protein X) is a multifunctional protein possessing ribonuclease activity (Rosenberg, 2008). It is known to be a marker of eosinophil presence and degranulation, and its elevated amounts in stool samples were reported to correlate with allergic reactions (Majamaa et al, 1999; Magnusson et al, 2003). Although some authors described elevated EDN in stool being associated with the presence of inflammation (Bischoff et al, 1997; Saitoh et al, 1999; Peterson et al, 2002; Wagner et al, 2008), these observations were inconclusive. Increased EDN values were also reported in colorectal perfusion fluid (Carlson et al, 1999) and material collected from the surface of the rectal mucosa using an inflatable intrarectal device (Anderson et al, 2011; the collecting device was described in US Pat. App. 2008/0097238). The latter two studies, however, assessed very few IBD cases. In a patent by Gleich and Levy (U.S. Pat. No. 5,928,883) EDN was proposed as one of eosinopil granule proteins (alongside eosinophil peroxidase), combined determination of which in whole gut lavage liquid could be used for IBD diagnosis. On the basis of the existing published evidence faecal EDN was not regarded as a promising biomarker of IBD, being less reliable than calprotectin or other stool biomarkers (Wagner et al, 2008; Foell et al, 2009). EDN was never considered as an IBD biomarker suitable for clinical use.
Several inflammatory cytokines were also proposed as potential biomarkers of IBD (Foell et al, 2009). Tumour necrosis factor alpha (TNFα), a small peptide predominantly produced by activated macrophages, could be a very good candidate, being a recognised therapeutic target in IBD patients (Danese et al, 2013). Although immunoassays for TNFα exist (e.g. U.S. Pat. Nos. 5,223,395; 5,436,154; 7,285,269), the protein is unstable in stool samples (Foell et al, 2009). This constitutes a serious obstacle for using it for diagnostic purposes.
Additional biomarkers that are not derived from inflammatory cells can also be informative in the context of IBD diagnosis and monitoring. For example it is generally accepted that cell adhesion molecules (CAMs) are closely involved in leukocyte trafficking constituting a major mechanism in inflammatory process (Springer, 1995). Among them, intercellular adhesion molecule-1 (ICAM-1) is known to play an especially important role in the development of IBD inflammatory bowel disease (Vainer, 2010). Detection of a common polymorphism in the gene encoding ICAM-1 appears to correlate with IBD risk and was previously proposed as an approach to genetic screening for predisposition to IBD development (U.S. Pat. Nos. 5,681,699; 6,008,335; 6,884,590). In addition, specific inhibition of ICAM-1 expression has recently been proposed as an approach to IBD treatment (Miner et al, 2006; Vainer, 2010). All available information regarding ICAM-1 presence in the colonic mucosa is limited by descriptive morphological observations from biopsy samples (Vainer, 2010), whereas it has never been quantified in either mucosal or stool samples.
Assessment of the degree of epithelial damage and its healing during convalescence constitutes another important aspect in the context of IBD. Soluble cytokeratin 18 (CK-18) is known to be released from epithelial cells following their death (Ueno et al, 2005). Although the presence of CK-18 in stool samples has never been investigated, elevated levels of this protein were once reported in samples obtained intrarectally from a few IBD patients (Anderson et al, 2011).
D-dimer is a small protein fragment generated during cross-linked fibrin degradation (Pabinger & Ay, 2009). Its increased presence can indicate chronic bleeding that is a common phenomenon in many IBD patients. Increased D-dimer levels in plasma samples from IBD patients were previously observed (Kume et al, 2007), but little was known on D-dimer changes in the gut. This biomarker could also be measured in intrarectally collected material (Anderson et al, 2011). D-dimer measurement might be informative for assessing intestinal inflammation severity and bleeding-related complication risk.
Some authors proposed using measurements of total human DNA in stool for IBD monitoring (Casellas et al, 2007), however the efficiency of this approach needs further evaluation. Finally, patent literature search allowed identifying less promising stool tests based upon the determination of intestinal 0-glycans (US Pat. App. No. 2009/0311707), HMGB1 protein (US Pat. App. No. 2013/0137123) and COX-2 protein (U.S. Pat. No. 7,220,825).
The presented background information shows that despite the availability of a number of potentially promising biomarkers of intestinal inflammation the field is still poorly developed. The only clinically employed biomarker-based test for IBD is stool calprotectin detection, the applicability of which is considerably limited due to the necessity of stool collection and handling. Presently there is no reliable alternative non-invasive test for IBD.
We have previously devised a new method of non-invasive collection of excreted colonic mucocellular layer (Loktionov, 2007) material from the anal area following natural bowel opening (WO2012/150453). This simple procedure based on sample self-collection provides material containing highly informative cells in abundance and can be easily applied to a range of biomarker detection-based diagnostic and monitoring applications in the area of colorectal disease. We have applied the new collection technique and tested a range of potential biomarkers in samples obtained from IBD patients and controls.
For convenience, a list of references cited herein follows: